Project Summary Clostridium perfringens type F strains, which produce C. perfringens enterotoxin (CPE), are an important cause of gastrointestinal (GI) disease, including the 2nd most common bacterial foodborne illness and several nonfoodborne GI diseases. In people with preexisting severe constipation or fecal impaction, type F infections can progress to lethal enterotoxemia, where CPE is absorbed from the intestines to damage organs such as the liver. CPE is produced when type F strains are ingested and then sporulate in the GI tract. Cytotoxicity starts with binding of CPE to claudin receptors to form a small complex. Six CPE small complexes then oligomerize on the plasma membrane of intestinal cells to form a surface prepore. Each of the six CPE molecules in the prepore extends a beta hairpin that inserts into the lipid bilayer to form a pore in the host cell plasma membrane. In vitro, this pore triggers a calcium influx that activates calpain and induces (at low CPE concentrations) apoptosis or (at high CPE concentrations) necroptosis. CPE-induced cell death and/or CPE effects on tight junctions (TJs) then cause intestinal damage that induces luminal fluid accumulation in the small intestine and colon. Therapeutics against CPE-mediated type F GI disease could target either CPE action or production during in vivo sporulation. For those efforts, or to improve use of CPE for translational applications such as cancer ther- apy, it is necessary to better understand CPE action at the molecular and intestinal levels and to improve know- ledge of early steps in C. perfringens sporulation and CPE production/processing in the intestines. Consequently, this project will pursue 4 specific aims: Aim 1, CPE has a dual action, i.e., pore formation and TJ damage, so we will evaluate if pore formation is necessary for CPE in vivo effects. A CPE point variant that binds and oligo- merizes but does not form an active pore will be used to test the importance of pore formation when CPE causes paracellular permeability effects on Caco-2 cells or enteritis and enterotoxemic death in animal models. Aim 2 will determine the importance/mechanism of indirect damage caused by CPE in vitro and in vivo. CPE binds mainly to villus tip cells yet damages the entire intestinal villus, supporting the involvement of indirect damage during CPE action in the intestines. Aim 2 will characterize a factor involved in CPE-induced bystander killing of Caco-2 cells and test if this factor is active in the intestines. This Aim will also evaluate if a similar indirect killing effect occurs in the CPE-treated intestines and if cytokine release contributes to intestinal damage. Aim 3 will use CPE variants to further probe the CPE structure/function relationship. Molecular events in CPE action to be examined include i) oligomerization, ii) contributions of a proline residue at the interface of the two CPE domains to CPE action, and iii) interactions between CPE and the 1st extracellular domain of claudin receptors. Aim 4 will study CPE processing and the regulation of sporulation/CPE production ex vivo using mouse intestinal lumen contents and test if bile salts (or other lumen factors) initiate type F strain sporulation/CPE production.